PROJECT SUMMARY This is a new proposal aimed at characterizing a novel mitochondrial pathway of interferon (IFN) signaling we discovered and to probe its relevance to autoimmune disease pathogenesis. IFNs are essential cytokines for host defense because they provoke intrinsic antiviral states in non-immune cells and stimulate immune cells and inflammation to combat infection. They are used therapeutically for certain autoimmune diseases, cancers, and viral infection, but are also implicated in pathology when produced chronically or out of context. In our studies of a Tfam+/- mouse model of mitochondrial DNA (mtDNA) stress and normal mitochondrial responses to viral infection, we found that mtDNA release into the cytoplasm primes IFN signaling to provide enhanced antiviral immunity. This response requires binding of mtDNA to the cytoplasmic nucleic acid sensor cGAS, activation of the endoplasmic reticulum (ER)-resident protein STING, and downstream signaling to up-regulate interferon-stimulated genes (ISGs) and Type 1 IFN production. ISG expression and increased Type 1 IFN signaling contribute to the autoimmune disease systemic lupus erythematosus (SLE or lupus). Our experimental plan will systematically interrogate key remaining questions regarding this new innate immune signaling pathway, including how mtDNA is released and recognized by cGAS, are altered mitochondrial dynamics and autophagy involved, and is the pathway relevant to human diseases states where mtDNA stress has been implicated. The specific aims of the project are to 1) determine the specificity and mechanism of mtDNA sensing by cGAS, 2) decipher the role of mitochondrial dynamics (fission, fusion, autophagy) in mtDNA release and IFN signaling, including development of an innovative new mtDNA-release assay, and 3) test if mtDNA stress-mediated IFN signaling contributes to lupus pathology in mouse models and human patient cells. Significance: Completion of this project will provide significant new insight into how mtDNA stress, which is observed in many disease conditions and aging, results in innate immune pathway activation and can lead to autoimmune pathology via increased IFN production, even in the absence of a viral infection. By defining the molecular mechanisms involved, new inroads into how to augment this response to enhance antiviral immunity or prevent it under pathological circumstances (e.g. in lupus) will be made that have therapeutic potential. Furthermore, we will gain considerable new basic biology insight into mitochondrial dynamics, cytoplasmic nucleic acid recognition pathways, autophagy, and mechanisms of mtDNA damage, repair and modification.